Methods and systems for polynucleotide detection

ABSTRACT

Optimization techniques for selecting indicator polynucleotides for an experiment and for determining expression levels resulting from the experiment. The optimization technique corrects for variations in polynucleotide melting temperatures during analysis of the experimental results. The optimization technique selects set of indicator polynucleotides for the experiment. The optimization technique then performs the experiment with the indicator polynucleotides and a sample and identifies the relative amounts of the indicated polynucleotides. The optimization technique then adjusts the relative amounts of the indicated polynucleotides based on melting temperatures associated with the indicator polynucleotides.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. patent application Ser.No. 10/146,720, entitled A METHOD TO ASSEMBLE SPLICE VARIANTS FOR BOTHKNOWN AND PREDICTED GENES, A METHOD FOR VALIDATING THEIR EXPRESSION INCELLS AND A METHOD OF DISCOVERING NOVEL EXON/EXON EXTENSIONS/TRIMS,filed May 14, 2002, which claims the benefit of U.S. Provisional PatentApplication No. 60/307,911, entitled A METHOD TO ASSEMBLE SPLICEVARIANTS FOR BOTH KNOWN AND PREDICTED GENES, A METHOD FOR VALIDATINGTHEIR EXPRESSION IN CELLS AND A METHOD OF DISCOVERING NOVEL EXON/EXONEXTENSIONS/TRIMS, filed Jul. 25, 2001; U.S. Patent Application No.60/343,298, entitled METHODS OF OLIGO SELECTION AND OPTIMIZATION, filedDec. 21, 2001; and U.S. Patent Application No. 60/329,914, entitled AMETHOD TO ASSEMBLE SPLICE VARIANTS FOR BOTH KNOWN AND PREDICTED GENES, AMETHOD FOR VALIDATING THEIR EXPRESSION IN CELLS AND A METHOD OFDISCOVERING NOVEL EXON/EXON EXTENSIONS/TRIMS, filed Oct. 17, 2001, whichare hereby incorporated by reference in their entirety.

TECHNICAL FIELD

[0002] The described technology relates generally to selecting indicatorpolynucleotides and to detecting polynucleotides.

BACKGROUND

[0003] Polymerase chain reaction (PCR) analyses, nucleotide arrayexperiments, in situ hybridizations, and Southern, Northern and Dot blotexperiments attempt to form DNA-DNA, RNA-RNA, or DNA-RNA hybrids. Insuch experiments, an “indicator polynucleotide,” such as anoligonucleotide probe, hybridizes to a polynucleotide that includes apolynucleotide subsequence complementary to the indicatorpolynucleotide. The “melting temperature,” which depends in part uponthe nucleotide sequence of the indicator polynucleotide, characterizesthe stability of the hybridization product given a set of experimentalconditions. The melting temperature is the temperature at which 50% of agiven indicator polynucleotide hybridizes to complementarypolynucleotides of sufficient abundance. The melting temperature iscritical for determining the selectivity and sensitivity of indicatorpolynucleotides when used as primers in polymerase chain reaction (PCR)experiments, as probes for in situ hybridizations, as probes fornucleotide array experiments, and in Southern, Northern, or Dot blotexperiments. If the melting temperature is too low, few indicatorpolynucleotides will hybridize to their complementary polynucleotides.If the melting temperature is too high, indicator polynucleotides mayhybridize to polynucleotides weakly homologous to their complementarypolynucleotides. Even with an optimal melting temperature, the formationof hybridization products may be influenced by experimental conditionsand the nucleotide sequences of the indicator polynucleotide and thepolynucleotides present in the biological sample or environment. Itwould be desirable to select indicator polynucleotides to maximizehybridization to complementary polynucleotides while minimizinghybridization to other polynucleotides. Also, it would be desirable ifthe post-hybridization analysis would factor in expected variations dueto differing melting temperatures of indicator polynucleotides as wellas expected variations due to homologous polynucleotides and otherpolynucleotides present in the sample or environment.

BRIEF DESCRIPTION OF THE DRAWINGS

[0004]FIG. 1 is a flow diagram illustrating the overall process ofselecting indicator polynucleotides to minimize the errors between theactual melting temperatures and the desired melting temperature in oneembodiment.

[0005]FIG. 2 is a flow diagram illustrating the overall process ofanalytically correcting for variations in melting temperatures in oneembodiment.

[0006]FIG. 3 is a flow diagram illustrating the overall process ofcorrecting for hybridization of an indicator polynucleotide with ahomologue of the target polynucleotide in one embodiment.

DETAILED DESCRIPTION

[0007] Optimization techniques for selecting indicator polynucleotidesfor an experiment and for determining expression levels resulting fromthe experiment are provided. In one embodiment, the optimizationtechnique corrects for variations in polynucleotide melting temperaturesduring analysis of the experimental results. The optimization techniqueselects set of indicator polynucleotides for the experiment. Theoptimization technique then performs the experiment with the indicatorpolynucleotides and a sample and identifies the relative amounts of theindicated polynucleotides. The optimization technique then adjusts therelative amounts of the indicated polynucleotides based on meltingtemperatures associated with the indicator polynucleotides. For example,if one of the indicator polynucleotides has a high melting temperaturerelative to the hybridization and wash temperatures, then theoptimization technique may increase the relative amount of thecorresponding indicated polynucleotide to account for the high meltingtemperature. In an alternate embodiment, the optimization techniqueincludes a control indicator polynucleotide to identify relative amountsof a homologue polynucleotide whose presence is incidentally detected byan indicator polynucleotide designed to detect the presence of a targetpolynucleotide.

[0008] In another embodiment, the optimization technique attempts tominimize the difference between the actual melting temperature of anindicator polynucleotide and a desired melting temperature. Whenmultiple indicator polynucleotides are used in a single experiment, theoptimization technique attempts to minimize the overall error resultingfrom differences in the melting temperatures of the indicatorpolynucleotides and the desired melting temperature. The optimizationtechnique may modify indicator polynucleotides prior to performing anexperiment to reduce the error. The optimization technique may modify anindicator polynucleotide by shifting the location of an indicatorpolynucleotide to hybridize with upstream or downstream portions of thetarget polynucleotide. The optimization technique may also vary thelengths of the indicator polynucleotides to minimize the error. Whendetecting exon-exon junctions, the optimization technique may attempt tobalance the melting temperature for each exon portion of the indicatorpolynucleotide so that the indicator polynucleotide will hybridize toboth exons equally. These and another optimization techniques aredescribed more fully in the following.

[0009] Measuring Error in Melting Temperatures

[0010] In one embodiment, the optimization technique selects anindicator polynucleotide from among multiple possibilities that willminimize an error equation, such as the following distance equation:

E ²=(T _(d) −T _(m))²  (1)

[0011] where E is the error, T_(d) is the desired melting temperature,and T_(m) is the theoretical or empirical melting temperature of thepolynucleotide. Equation 1 is generally referred to as an Euclideandistance measure. The indicator polynucleotide with the smallest errormay be the best choice.

[0012] When multiple indicator polynucleotides are to be used in thesame experiment, the optimization technique selects indicatorpolynucleotides that tend to minimize the overall error in meltingtemperatures. In one embodiment, the optimization technique calculatesthe error according to the following equation.

E _(t) ² =E ₁ ² +E ₂ ² + . . . +E _(n) ²  (2)

[0013] where E_(t) is the total error, E_(l) is the error from the ithpolynucleotide, and n is the number of indicator polynucleotides.

[0014] A more general formulation of error equation 1 that applies to asingle polynucleotide is the following equation:

E=f(T _(d) , T _(m))  (3)

[0015] where f is any arbitrary error function. The substitution ofequation 3 into equation 2 for multiple polynucleotides results in thefollowing equation:

_(t) =g(f(T _(d) , T _(m1)), f(T _(d) , T _(m2)), . . . , f(T _(d) , T_(mn)))  (4)

[0016] where g is a function that combines the individual error measuresof the n indicator polynucleotides. When an experimental design includesconstraints, such as which genes, exons, or exon-exon junctions theindicator polynucleotides identify, the optimization technique selectsthose indicator polynucleotides that minimize the value of E_(t) giventhe desired melting temperature T_(d).

[0017] To illustrate the error calculation, an example nucleotide arrayexperiment involving indicator polynucleotides for each exon andexon-exon junction of a transcript of the gene CD44 with the GenBanklocus name XM_(—)030326, which contains 18 exons and 17 exon-exonjunctions, is used. One indicator polynucleotide selection techniquemight select indicator polynucleotides with a length of 20 bases. Eachindicator polynucleotide for an exon is selected to hybridize to thecenter of the exon, and each indicator polynucleotide for an exon-exonjunction is selected to hybridize to 10 bases on each side of theexon-exon junction. The selected indicator polynucleotides are presentedin the following table: TABLE 1 Junction/ Exon Melting Temp ° C.Sequence E1 62.030003 cgcgcccagggatcctccag J1-2 22.0gcgcagatcgatttgaatat E2 59.980003 ggaggccgctgacctctgca J2-3 30.0agacctgcaggtatgggttc E3 53.83 tgcagcaaacaacacagggg J3-4 32.0aatgcttcagctccacctga E4 53.83 agtcacagacctgcccaatg J4-5 24.0attaccataactattgttaa E5 55.880005 gctcctccagtgaaaggagc J5-6 28.0cctgctaccactttgatgag E6 51.780003 cctgggattggttttcatgg J6-7 30.0caaatggctggtacgtcttc E7 49.730003 gatgaaagagacagacacct J7-8 28.0tccagcaccatttcaaccac E8 55.880005 tggacccagtggaacccaag J8-9 28.0aggatgactgatgtagacag E9 53.83 aagcacaccctcccctcatt  J9-10 32.0acaagcacaatccaggcaac E10 51.780003 ggtttggcaacagatggcat J10-11 34.0gggacagctgcagcctcagc E11 49.730003 gacagttcctggactgattt J11-12 28.0agaaggatggatatggactc E12 47.68 aatccaaacacaggtttggt J12-13 28.0atgacaacgcagcagagtaa E13 47.68 gaaggcttggaagaagataa J13-14 26.0acatcaagcaataggaatga E14 51.780003 acgaaggaaagcaggacctt J14-15 30.0tccttatcaggagaccaaga E15 57.930004 cagtggggggtcccatacca J15-16 30.0gaatcagatggacactcaca E16 53.83 ggagcaaacacaacctctgg J16-17 28.0caaattccagaatggctgat E17 49.730003 ccttggctttgattcttgca J17-18 34.0gtcgaagaaggtgtgggcag E18 53.83 tcgttccagttcccacttgg

[0018] The NCBI locus name and version for the genomic sequencecontaining the CD44 gene is NT_(—)024229.8. The NCBI locus name andversion for the mRNA sequence of the CD44 transcript is XM_(—)030326.3.The identifiers E1, E2, E3, and etc. identify indicator polynucleotidesfor exon 1, exon 2, exon 3, and etc. The identifiers J1-2, J2-3, andetc. identify indicator polynucleotides for the exon-exon junctionbetween exon 1 and exon 2, the exon-exon junction between exon 2 andexon 3, and etc. The melting temperatures are theoretical meltingtemperatures of the indicator polynucleotide, calculated according tothe following equation:

T _(m)=64.9° C.+41° C.*(GC−16.4)/L  (5)

[0019] where L is the length of the target polynucleotide and GC is theGC content. One skilled in the art will appreciate the theoreticalmelting temperature can be calculated using various well-knownequations. The calculated melting temperature varies from a low of 22°C. to a high of 62.03° C. in this example. This wide range of meltingtemperatures will lead to substantial variation in the number ofpolynucleotides that hybridize to instances of a given indicatorpolynucleotides in an experiment with fixed hybridization and washingtemperatures. For example, in an experiment with a sample containingCD44 using a standard protocol with a hybridization temperature of 52°C. and a washing temperature of 52° C., the indicator polynucleotideswith melting temperatures under 52° C. will form and retainhybridization products less frequently than those with meltingtemperatures above 52° C. In general, indicator polynucleotides with lowcalculated melting temperatures may not bind to their target exons orexon-exon junctions with any measurable strength above a backgroundlevel. The error with a target temperature of 52° C. is calculated usingequation 2 in the following:

E _(t) ²=(52−62.03)²+(52−22)²+ . . . +(52−53.83)²=(97.46)²

[0020] Modifying Indicator Polynucleotides to Reduce Errors in MeltingTemperatures

[0021] The optimization technique can optimize an indicatorpolynucleotide prior to running the experiment. The optimizationtechnique may optimize indicator polynucleotides by shifting thelocation of the indicator polynucleotides slightly upstream ordownstream within their respective RNAs. For example, the optimizationtechnique can select indicator polynucleotides for exons from any baserange within the exon that the indicator polynucleotide identifies,using any selection criteria, such as GC content. Alternatively, theoptimization technique can optimize indicator polynucleotides by varyingtheir lengths. For example, rather than selecting only indicatorpolynucleotides with a length of 20 bases, the optimization techniquemay select indicator polynucleotides of varying lengths to reduce theerror measure for each indicator polynucleotide. These variousoptimizations may be used alone or in conjunction with other indicatorpolynucleotide selection, analytical correction, or other optimizations.When varying the length of the indicator polynucleotides for the sameCD44 transcript, without using other indicator polynucleotide selectionor optimizations, the resulting indicator polynucleotide are shown inthe following: TABLE 2 Junction/ Melting Exon Temp ° C. Sequence E151.062504 gcccagggatcctcca J1-2 52.258335 cgcagatcgatttgaatataacct E251.062504 aggccgctgacctctg J2-3 53.24737 gacctgcaggtatgggttc E351.089478 tgcagcaaacaacacaggg J3-4 51.089478 aatgcttcagctccacctg E451.089478 agtcacagacctgcccaat J4-5 51.653847 caattaccataactattgttaaccgtE5 53.24737 ctcctccagtgaaaggagc J5-6 51.780003 cctgctaccactttgatgag E652.97273 cctgggattggttttcatggtt J6-7 51.780003 caaatggctggtacgtcttc E751.109093 aagatgaaagagacagacacct J7-8 51.780003 ccagcaccatttcaaccaca E852.600002 ggacccagtggaacccaa J8-9 51.7087 aaggatgactgatgtagacagaa E951.876472 gcacaccctcccctcat  J9-10 52.404762 tacaagcacaatccaggcaac E1051.780003 ggtttggcaacagatggeat J10-11 51.876472 ggacagctgcagcctca E1152.404762 gacagttcctggactgatttc J11-12 52.97273 aagaaggatggatatggactccE12 51.109093 caaatccaaacacaggtttggt J12-13 51.7087aatgacaacgcagcagagtaatt E13 51.7087 gaaggcttggaagaagataaaga J13-1451.7087 gacatcaagcaataggaatgatg E14 51.089478 acgaaggaaagcaggacct J14-1550.452385 ttccttatcaggagaccaaga E15 51.062504 tggggggtcccatacc J15-1651.109093 tgaatcagatggacactcacat E16 51.089478 ggagcaaacacaacctctgJ16-17 52.97273 ccaaattccagaatggctgatc E17 52.404762ccttggctttgattcttgcag J17-18 52.600002 gtcgaagaaggtgtgggc E18 51.089478tcgttccagttcccacttg

[0022] Since the calculated polynucleotide melting temperatures have asmall range, from 50.45° C. to 52.97° C. (rather than 22° C. to 62.03°C.), the error measure Et of 4.51 (rather than 97.46) is also small.This varying of the lengths of the indicator polynucleotides haseliminated over 95% of the error encountered when the indicatorpolynucleotides were selected as shown in Table 1.

[0023]FIG. 1 is a flow diagram illustrating the overall process ofselecting indicator polynucleotides to minimize the errors between theactual melting temperatures and the desired melting temperature in oneembodiment. In block 101, the technique selects an initial set ofindicator polynucleotides. In block 102, the technique inputs thedesired melting temperature of the indicator polynucleotides. In blocks103-105, the technique modifies each indicator polynucleotide tominimize its error. In block 103, the technique selects the nextindicator polynucleotide. In decision block 104, if all the indicatorpolynucleotides have already been selected, then the techniquecompletes, else the technique continues at block 105. In decision block105, if the error between actual melting temperature of the selectedindicator polynucleotide and the desired melting temperature isacceptable, then the technique selects the next indicatorpolynucleotide, else the technique continues at block 106. In block 106,the technique modifies the selected indicator polynucleotide using oneor more of the described techniques (e.g., adjusting the length of theindicator polynucleotide or moving the indicator polynucleotide upstreamor downstream) and then continues at a block 105.

[0024] One skilled in the art will recognize that the error Et may befurther reduced in a variety of ways, such as by introducing moleculesother than the bases A, C, G, or T with different bindingcharacteristics. Such bases may be appended, prepended, inserted, orselectively substituted within the indicator polynucleotides.

[0025] CD44 is known to have several splice variants. For example, thetranscript used above contains several exons not present in other spliceforms of CD44. In particular, J6-7 does not exist in some species ofCD44. Instead, E6 joins with a later exon E7′, yielding J6-7′. The RNAtranscript containing the J6-7 is referred to as R¹, and the RNAtranscript containing J6-7′ is referred to as R².

[0026] When using indicator polynucleotides to identify exon-exonjunctions, binding may be less specific than desired if polynucleotidesbind to one or the other half of an indicator polynucleotide. Forexample, if J6-7 is not present in a given sample, but E6 is present(because a different splice form of the gene is present in that sample),then an indicator polynucleotide for J6-7 may hybridize to E6 eventhough E6 is not joined to E7 in the splice variant in the sample. Thehybridization, however, will likely be weaker than if a splice variantcontaining J6-7 was present. The expression levels may be measured as

H(E6)=1069

H(J6-7)=388

[0027] where H(E6) is the measured expression level of E6 in theexperiment and H(J6-7) is the measured expression level of J6-7. Thesemeasured expression may represent the following scenarios:

[0028] 1. R¹ is present but R² is not present.

[0029] 2. R¹ and R² are both present.

[0030] 3. Neither R¹ nor R² is present, but another splice variant R³ ispresent. R³ contains both E6 and E7, but not J6-7, because somealternate splicing event or events occurs between E6 and E7.

[0031] Techniques described in U.S. patent application Ser. No.10/146,720, entitled “Method and System for Identifying Splice Variantsof a Gene,” can be used to differentiate these scenarios. Thosetechniques assign an expected expression level to J6-7 in the presenceof R¹, in the presence of R², and in the presence of R³. For example, ifthere are indicator polynucleotides for E6, E7, and J6-7, then a matrixM with a column for each expected splice variant (e.g., R¹, R² and R³)and a row for each indicator polynucleotide (e.g., E6, E7, and J6-7) iscreated. The values in the matrix correspond to the expected expressionlevel for the target polynucleotides. When the partial expression levelsare not expected, the matrix might-look like the following:$\begin{matrix}\quad & \quad & R^{1} & R^{2} & R^{3}\end{matrix}$ $M = {\begin{matrix}{1\quad} & {\quad 1} & {\quad 1} \\{1\quad} & {\quad 0} & {\quad 0} \\{1\quad} & {\quad 0} & {\quad 1}\end{matrix}\begin{matrix}\begin{matrix}{E6} \\{{J6}\text{-}7}\end{matrix} \\{E7}\end{matrix}}$

[0032] If the indicator polynucleotide for J6-7, however, weakly bindsin the presence of a different splice site, J6-7′, a correction can beapplied. The values in the correction matrix can be calculated orempirically derived. The values can be empirically derived by performinga hybridization experiment containing J6-7′ but not J6-7. The correctionmatrix may be derived using one or more samples containing antisensepolynucleotides. A sample could include antisense polynucleotides for J6or J7 or both. (“J6” refers to the portion of an indicatorpolynucleotide for a J6-X junction that is used to identify the E6portion of the junction.) For example, an antisense polynucleotide forJ6 might contain the complementary polynucleotide for J6. Alternatively,the antisense polynucleotide might contain the complementarypolynucleotide for J6 appended to a sequence of some additional numberof bases, perhaps chosen randomly. In yet another scenario, theantisense polynucleotide might contain J6 with J7 prepended. After thehybridization experiments, the following expression values may result:

H(J6-7|J6)=556

H(J6-7|J7)=310

H(J6-7|J6-7)=1544

H(J6-7|J6, J7)=756

[0033] where H(J6-7|J6) is the empirically derived expression level ofthe indicator polynucleotide for J6-7 in the presence of a samplecontaining antisense polynucleotides for J6, H(J6-7|J7) is theexpression level of J6-7 in the presence of a sample containingantisense polynucleotides for J7, H(J6-7|J6-7) is the ordinaryexpression level of J6-7 in the presence of antisense polynucleotidesfor J6-7, and H(J6-7|J6, J7) is the expression level of J6-7 in thepresence of separate antisense polynucleotides for J6 and J7. Theexpression values are not independent and are preferably measured inseparate hybridization experiments. If more than one expression level ismeasured in the same experiment, the individual values can be solvedusing a system of linear equations, a least squares equation, or anotherdeconvolution method.

[0034] Once the expression levels have been determined eitherempirically or theoretically, the values in the matrix M could bedetermined using an equation such as:

M _(l,j) =H(P _(i) |R _(j))/H(P _(i))  (6)

[0035] where M_(l,j) is a coefficient matrix, P_(i) is an indicatorpolynucleotide that identifies a subsequence of RNA transcript R_(j),H(P_(i)|R_(j)) is the expression level of indicator polynucleotide P_(i)given that RNA R_(j) is expressed, and H(P_(i)) is the expression levelof the hybridization product of indicator polynucleotide i. The solutionto this equation is:

M _(2,1) =H(J6-7|J6-7)/H(J6-7|J6-7)=1544/1544−1=1

M _(2,2) =H(J6-7|J6)/H(J6-7|J6-7)=556/1544=0.36

M _(2,3) =H(J6-7|J6,J7)/H(J6-7|J6-7)=756/1544=0.49

[0036] In this example, H(J6-7|J6-7) is the expression of the indicatorpolynucleotide for J6-7 given RNA containing J6-7 is present. Theremaining matrix elements all have a value of 1, for the same reason asM_(2,1). The resulting matrix M is: $\begin{matrix}\quad & \quad & {R^{1}\quad} & R^{2} & {\quad R^{3}}\end{matrix}$ $M = {\begin{matrix}{1\quad} & {\quad 1} & 1 \\{1\quad} & {\quad 0.36} & 0.49 \\{1\quad} & {\quad 0} & 1\end{matrix}\begin{matrix}\begin{matrix}{E6} \\{{J6}\text{-}7}\end{matrix} \\{E7}\end{matrix}}$

[0037] The value 0.36 indicates that the indicator polynucleotide forJ6-7 will yield a relative expression level of 0.36 times the full valueif R² is present, and the other two splice variants are not present. Theexpression level is non-zero in this case because the indicatorpolynucleotide for J6-7 will hybridize weakly in the presence of E6 evenif J6-7 is not present. The value 0.49 indicates that the indicatorpolynucleotide for J6-7 will yield a relative expression level of 0.49times the full value if R³ is present and the other two splice variantsare not present. The value for J6-7 in R³ is larger than thecorresponding value in R² because the indicator polynucleotide willhybridize weakly to both E6 and E7 rather than only to E6.

[0038] Modifying a Junction Indicator Polynucleotide to Balance ItsMelting Temperature

[0039] The indicator polynucleotide for J6-7 consists of a portion J6that identifies the 3′ end of E6 as well as a portion J7 that identifiesthe 5′ end of E7. Each of these portions has its own meltingtemperature. In other words, the indicator polynucleotide for J6-7 willhybridize to E6 based on the melting temperature of J6 even if J6-7 isnot present in the sample. Likewise, the indicator polynucleotide willhybridize to E7 based on the melting temperature of J7 even if J6-7 isnot present in the sample. In one embodiment, the optimization techniquebalances the melting temperature of each exon portion of a junctionindicator polynucleotide.

[0040] A full set of exon-exon junction indicator polynucleotides with alength of 30 bases for CD44 selected to have 15 bases for each exon isshown in the following: TABLE 3 Junction/ Melting Left Melting RightMelting Exon Temp ° C. Temp ° C. Temp ° C. Sequence J1-2 61.62000350.140003 28.273338 gcctggcgcagatcgatttgaatataacct J2-3 60.25333439.20667 36.473335 atttgagacctgcaggtatgggttcataga J3-4 62.98666839.20667 41.940002 gcttcaatgcttcagctccacctgaagaag J4-5 57.520004 33.7436.473335 gaccaattaccataactattgttaaccgtg J5-6 61.620003 41.94000236.473335 gaatccctgctaccactttgatgagcacta J6-7 58.88667 39.20667 33.74caacacaaatggctggtacgtcttcaaata J7-8 61.620003 39.20667 39.20667ttatctccagcaccatttcaaccacaccac J8-9 60.253334 41.940002 33.74ccacaaggatgactgatgtagacagaaatg  J9-10 60.253334 33.74 41.940002attctacaagcacaatccaggcaactccta J10-11 65.72 44.673336 41.940002caacagggacagctgcagcctcagctcata J11-12 62.986668 41.940002 39.20667caggaagaaggatggatatggactccagtc J12-13 58.88667 36.473335 36.473335tttcaatgacaacgcagcagagtaattctc J13-14 58.88667 39.20667 33.74ctctgacatcaagcaataggaatgatgtca J14-15 60.253334 36.473335 39.20667atcgttccttatcaggagaccaagacacat J15-16 61.620003 36.473335 41.940002gatctgaatcagatggacactcacatggga J16-17 61.620003 41.940002 36.473335caccccaaattccagaatggctgatcatct J17-18 62.986668 39.20667 41.940002caacagtcgaagaaggtgtgggcagaagaa

[0041] The melting temperature is the calculated melting temperature ofthe complete indicator polynucleotide, the left melting temperature isthe calculated melting temperature of the first 15 bases of theindicator polynucleotide which detect the 3′ end of the first exon ineach junction, and the right melting temperature is the calculatedmelting temperature of the last 15 bases of the indicator polynucleotidewhich detect the 5′ end of the second exon in each junction. Thevariation between the two sides of a given indicator polynucleotide maybe considerable; the difference between the left and right meltingtemperature for the same indicator polynucleotide is as large as 21.87°C. for J1-2.

[0042] The temperature difference in J6-7 is approximately 5.5° C. Theexpected result of the difference is that the indicator polynucleotidewill yield a larger expression value if E6 is present without J6-7 thanif E7 is present without J6-7. A similar effect will be observed tovarying degrees for all of the indicator polynucleotides. In oneembodiment, the optimization technique corrects for these effects usingthe linear equations presented above. For example, different values fora transcript containing E6 but not E7 than for a transcript containingE7 but not E6 may be used. The corrected matrix might look like this:

[0043] R¹ R² R³ $M^{c} = {\begin{matrix}1.00 & 1.00 & 0.00 \\1.00 & 0.41 & 0.29 \\1.00 & 0.00 & 1.00\end{matrix}\begin{matrix}\begin{matrix}{E6} \\{{J6}\text{-}7}\end{matrix} \\{J7}\end{matrix}}$

[0044] where R² is a transcript containing E6 but not E7 and R³ is atranscript containing E7 but not E6. However, it would be desirable tominimize the need to correct experimental results in this way, since itis generally desirable to minimize the number of experimental parametersthat vary in the same experiment. The optimization technique minimizesor eliminates the need for this correction by balancing the meltingtemperature on each side of the exon-exon junction, so that the meltingtemperatures of indicator polynucleotide portions J6 and J7 are close toequal.

[0045] An optimization equation for error from imbalanced meltingtemperature using Euclidean distance can be written as:

E _(x) ²=(T _(ia) −T _(ib))²  (7)

[0046] where E_(x) is the error from imbalance in melting temperature,T_(ia) is the calculated or empirical melting temperature of the portionof the indicator polynucleotide which identifies the 3′ end of the firstexon, and T_(ib) is the calculated melting temperature of the portion ofthe indicator polynucleotide which identifies the 5′ end of the secondexon. If the indicator polynucleotide does not identify an exon-exonjunction, the error is zero. The total error in the experiment then isgiven by:

E _(xt) ² =E _(x1) ² +E _(x2) ² + . . . +E _(xn) ²  (8)

[0047] where E_(xt) is the total error from multiple indicatorpolynucleotides as a result of temperature imbalance in indicatorpolynucleotides that identify exon-exon junctions. One skilled in theart will appreciate that error metrics other than Euclidean distance maybe used.

[0048] In one embodiment, the optimization technique considers bothtemperature balancing in exon-exon junction indicator polynucleotidesand total indicator polynucleotide melting temperature, which can berepresented by the following equation:

E _(jt) =k ₁ E+k ₂ E _(x)  (9)

[0049] where E_(jt) is the joint error measure, E is given by equation1, E_(x) is given by equation 7, and k₁ and k₂ are constants. The totaljoint error measure can be represented by the following equation:

E _(jt) =E _(j1) +E _(j2) + . . . +E _(jn)  (10)

[0050] where E_(jt) is the total error from both temperature imbalancein exon-exon junction indicator polynucleotides and each E_(ji) is theerror for each individual indicator polynucleotide i calculated usingequation 9. One skilled in the art will appreciate that error metricsother than Euclidea distance can be used and that equations 710 can begeneralized as equations 3 and 4 were generalized.

[0051] The exon-exon junction indicator polynucleotides selected fromCD44 according to equations 7-10, using a desired total meltingtemperature Td=60° C. and k₁=k₂=1.0 are shown in the following: TABLE 4Junction/ Melting Left Melting Right Melting Exon Temp ° C. Temp ° C.Temp ° C. Sequence J1-2 59.921432 38.0 37.405884ggcgcagatcgatttgaatataacctgc J2-3 59.921432 38.0 38.25tgagacctgcaggtatgggttcatagaa J3-4 61.255558 39.20667 38.0gcttcaatgcttcagctccacctgaag J4-5 59.59412 39.81765 39.81765tggaccaattaccataactattgttaaccgtgat J5-6 59.737038 37.37143 38.0aatccctgctaccactttgatgagcac J6-7 60.40323 39.20667 38.25caacacaaatggctggtacgtcttcaaatac J7-8 59.737038 37.37143 38.0tatctccagcaccatttcaaccacacc J8-9 60.253334 37.37143 38.25cacaaggatgactgatgtagacagaaatgg  J9-10 59.921432 38.25 38.0cattctacaagcacaatccaggcaactc J10-11 59.980003 34.0 34.0gggacagctgcagcctcagc J11-12 59.737038 37.37143 38.0aggaagaaggatggatatggactccag J12-13 58.88667 36.473335 36.473335tttcaatgacaacgcagcagagtaattctc J13-14 60.54375 39.20667 39.81765ctctgacatcaagcaataggaatgatgtcaca J14-15 59.737038 38.0 37.37143cgttccttatcaggagaccaagacaca J15-16 61.506897 40.8125 40.0ggatctgaatcagatggacactcacatgg J16-17 60.093105 37.37143 36.473335accccaaattccagaatggctgatcatct J17-18 59.324 38.0 38.0acagtcgaagaaggtgtgggcagaa

[0052] Variations between the left melting temperature and right meltingtemperature values has decreased significantly; the maximum differencebetween left melting temperature and right melting temperature is nowonly approximately 0.95° C. The temperature balancing technique haseliminated over 95% of the variability in melting temperature betweenthe two portions of each exon-exon junction indicator polynucleotide ascompared to the indicator polynucleotide selection technique. Themelting temperature of a fixed-length indicator polynucleotide can bebalanced by decreasing the length of one exon's portion and increasingthe length of the other exon's portion. For example, if the length ofthe indicator polynucleotide for a junction is 20 and the length of eachportion is 10, then the length of one portion may be decreased to 8 andthe length of the other portion might be increased to 12 to balance themelting temperature, keeping the overall length at 20. Corrections canbe applied to experimental results, as described above, to furtherreduce the variability.

[0053] Correcting Analytically for Variations in Melting Temperature

[0054] In one embodiment, the optimization technique corrects forvariations in polynucleotide melting temperatures during analysis of theexperimental results. This correction can be used whether or not anoptimal set of indicator polynucleotides is selected as described above.The optimization technique adjusts the detected expression levels of thepolynucleotides based on theoretical and desired melting temperatures.FIG. 2 is a flow diagram illustrating the overall process ofanalytically correcting for variations in melting temperatures in oneembodiment. The steps of the processes illustrated by the flow diagramsmay be controlled by, performed with the assistance of, or performed bya computer system. One skilled in the art will appreciate that varioussteps may be performed manually. In block 201, the technique selects theindicator polynucleotides that are to be used to identify targetpolynucleotides in a sample. In block 202, the technique performs thehybridization experiment, for example, using nucleotide arraytechnology. In block 203, the technique receives the resultingexpression levels from the experiment. In block 204, the techniquecalculates or inputs the melting temperature for each of the indicatorpolynucleotides. In block 205, the technique adjusts the expressionlevels based on the melting temperature for each of the indicatorpolynucleotides and temperature (e.g., hybridization temperature or washtemperature) at which the experiment was performed to give the finalcalculated expression levels for the experiment. One example analysistechnique is described in U.S. patent application Ser. No. 10,146,720,filed on May 14, 2002 and entitled “Method and System for IdentifyingSplice Variants of a Gene,” which is hereby incorporated by reference.That splice variants analysis calculates the expression levels accordingto the following equation:

S=MH  (11)

[0055] where S is a solution matrix of expression levels with a row foreach expected RNA transcript and a column for each experiment, M is amatrix of coefficients in which each column corresponds to an expectedRNA transcript and each row corresponds to an indicator polynucleotidein the experiment (each coefficient indicates the relative expectedexpression level of the indicator polynucleotide for the expected RNAtranscript), and H is a matrix in which each of the columns correspondsto expression values derived from an expression array experiment.Because of experimental noise, an exact solution to this equation maynot exist. The splice variants analysis can find an approximation usinga variety of Techniques such as a least squares regression using thefollowing equation:

S=(MM ^(T))⁻¹ M ^(T) H  (12)

[0056] where M^(T) is the transpose of M. The values in the coefficientmatrix M can be represented by the following equation:

M _(l,j) =L(P _(i) |R _(j))  (13)

[0057] whereM_(i,j is the matrix element in the ith row and jth column and L(P)_(i)|R_(j)) defines a coefficient for indicator polynucleotide P (in theith row of M) given that the expected RNA transcript R (in the jthcolumn of M) is present in a sample.

[0058] If all indicator polynucleotides that identify subsequences of Rare expected to be expressed at an identical level when R is present inthe sample, and all indicator polynucleotides that do not identifysubsequences of R are expected not to be expressed at all, matrix Mmight consist entirely of zeros and ones. The matrix M for two splicevariants of CD44, R¹ and R², is represented by the following:$\quad \begin{matrix}{{R^{1}\quad R^{2}}\quad} \\{M = {\begin{matrix}{\quad 1\quad} & 1 \\{\quad 1} & 1 \\{\quad 1} & 1 \\{\quad 1} & 1 \\{\quad 1} & 1 \\{\quad 1} & 1 \\{\quad 1} & 0 \\{\quad 1} & 0 \\{\quad 1} & 0 \\{\quad 1} & 0 \\{\quad 1} & 0 \\{\quad 1} & 0 \\{\quad 0} & 1 \\{\quad 1} & 0 \\{\quad 1} & 1 \\{\quad 1} & 1 \\{\quad 1} & 1 \\{\quad 1} & 1 \\{\quad 1} & 1 \\{\quad 1} & 1 \\{\quad 1} & 1 \\{\quad 1} & 1 \\{\quad 1} & 1 \\\quad & \quad\end{matrix}\begin{matrix}{E1} \\{E2} \\{E3} \\{E4} \\{E5} \\{E6} \\{{J6}\text{-}7} \\{E7} \\{{J7}\text{-}8} \\{E8} \\{{J8}\text{-}9} \\{E9} \\{{J6}\text{-}10} \\{{J9}\text{-}10} \\{E10} \\{E11} \\{E12} \\{E13} \\{E14} \\{E15} \\{E16} \\{E17} \\{E18}\end{matrix}}}\end{matrix}$

[0059] The value of 0 indicates the expected expression level will bezero if the target polynucleotide.g., J6-10) is not present in thesample. The uniform value of 1 indicates the expected expression levelsof all other indicator polynucleotides will be equal if the expected RNAtranscript is present in the sample. The indicator polynucleotides,however, may identify their target polynucleotides at varying expressionlevels because of variations in the melting temperature of the indicatorpolynucleotides.

[0060] In one embodiment, the optimization technique adjusts thedetected expression levels to account for the variations in meltingtemperatures. Continuing with the CD44 gene example, the calculatedmelting temperatures are Used to scale the expected expression levels inmatrix M. If the expected expression levels varied linearly with respectto temperature, then the expected expression levels can be adjustedaccording to the following equation:

M _(i,j) =T _(i) /T _(d)  (14)

[0061] where M_(i,j) is the element in the ith row and jth column of thematrix M, T_(i) is the expected melting temperature of the indicatorpolynucleotide corresponding to row i, and T_(d) is the desired meltingtemperature for use in the experiment. If the melting temperature is62.03 and the desired melting temperature is 52, then the adjustedexpression level would be 1.19 (i.e., 62.02/52).

[0062] Alternatively, the optimization technique performs the correctionby adjusting the detected expression levels of matrix H, rather thanadjusting the coefficients of matrix M. If the detected expression levelvaried linearly with respect to temperature, the detected expressionlevels can be adjusted according to the following equation:

H′ _(i,j) =H _(i,j) T _(d) /T _(I)  (15)

[0063] where H_(i,j) is the detected expression level for indicatorpolynucleotide i in experiment j, H′_(i,j) is the corrected expressionlevel, T_(i) is the expected melting temperature of the indicatorpolynucleotide corresponding to row i, and T_(d) is the desired meltingtemperature. If the melting temperature is 62.03, the desired meltingtemperature is 52, and the detected expression level is 11,869, then theadjusted expression level is 9950 (i.e., 11,869*52/62.03).

[0064] Since expected expression levels will likely vary with respect totemperature according to an equation that is more complex than a linearequation, the optimization technique in one embodiment may apply acorrection based on one or more empirical hybridization experiments. Onesuch experiment creates a sample consisting of synthesizedpolynucleotides to bind to indicator polynucleotides on a nucleotidearray. The optimization technique then calculates the expectedexpression levels of matrix M according to the following equation:

M _(i,j) =kH _(i)  (16)

[0065] where M_(i,j) is the element in the ith row and jth column of thematrix M, Hi is an empirically derived expression level for theindicator polynucleotide i from a hybridization experiment, and k is aconstant. For example, if the expression level of an indicatorpolynucleotide is 1481 and the average expression level from theexperiment is 1208, then k is {fraction (1/1208)} resulting in acorrected expected expression level of 1.23 (e.g., ({fraction(1/1208)})*1481).

[0066] The optimization technique can also correct for 3′ bias, whichoccurs in some methods of RNA amplification, such as T7 amplification.In one embodiment, the optimization technique calculates the correctedexpression level according to the following equation:

M _(i,j) =f(H _(i,b))  (17)

[0067] where f is a function such as a linear or nonlinear equation andH_(i,b) is the expected expression level for an indicator polynucleotidei which identifies a target polynucleotide located b bases from the 3′end of the RNA transcript in column j. For example, if the expressionlevel for polynucleotides drops off linearly measured by the number ofbases from the 3′ end of the RNA transcript, function f can berepresented by the following equation:

f(H _(i,b))=k ₁ −k ₂ b  (18)

[0068] where k₁ is the maximum value at the 3′ end of the polynucleotideand k₂ is the rate at which the expression level drops for eachnucleotide base from the 3′ end.

[0069] If k₁ is 1.00, k₂ is 1.2E-2, and the target polynucleotide islocated 1,303 bases from the 3′ end of the RNA transcript, then thevalue of function f is represented by the following equation:

M _(i,j) =f(H _(i, 1303))=1.00−1.2E-4*1303=0.84364

[0070] Alternatively, the optimization technique can apply the 3′ biascorrection to the empirically derived expression levels of matrix H,rather than matrix M, resulting in the following equation:

H′ _(1,1) =H _(i,j) f(H _(i,b))⁻¹=11,869*(0.84364)⁻¹=14,069

[0071] The optimization technique can use more complex functions whenthe expression level varies nonlinearly with respect to the distancefrom the 3′ end. For example, the expression level might dropprecipitously when b exceeds some value. The optimization technique canuse a nonlinear equation to represent such a drop.

[0072] The optimization technique can apply multiple corrections to thesame matrix as indicated by the following equation:

M′ _(i,j)=(M _(i,j) *C ¹ _(i,j) *C ² _(i,j) * . . . * C ^(n)_(i,j)  (19)

[0073] where M′ is the corrected matrix, M_(i,j) is a starting matrixelement value, and the various C_(i,j) are element-by-elementmultiplicative corrections such as the 3′ bias correction describedabove. The optimization technique can represent the correction moregenerally by the following equation:

M′ _(i,j) =f(M _(i,j) , C ¹ _(i,j) , C ² _(i,j) , . . . , C ^(n)_(i,j))  (20)

[0074] where the C_(i,j)s are correction factors that may be appliedusing element-by-element corrections, such as multiplication, addition,or subtraction. The optimization technique can alternatively apply themultiple correction factors to matrix H, rather than matrix M accordingto the following equations:

H′ _(i,j) =H _(i,j)*(C ¹ _(i,j))⁻¹*(C ² _(i,j))⁻¹* . . . *(C ^(n)_(i,j))⁻¹  (21)

H′ _(i,j) =f(H _(i,j) , C ¹ _(i,j) , C ² _(i,j) , . . . , C ^(n)_(i,j))  (22)

[0075] where f is a function that applies each individual correction tothe empirically derived expression level.

[0076] Correcting for Presence of Homologous Polynucleotides

[0077] In one embodiment, the optimization technique corrects for thepresence of homologous polynucleotides in a sample. In particular, anindicator polynucleotide identifies a target polynucleotide in an RNAtranscript, but it may also identify, albeit less strongly, a homologueof the target polynucleotide in a different RNA transcript. The RNAtranscripts may differ by only a few bases. The optimization techniquecan correct for a homologue in the sample by adding a column to matrix Mfor the homologue. The column might have a value only in row i if noother indicator polynucleotide in the experiment identifies asubsequence of the different RNA transcript. The solution can be foundin various ways, such as using a system of linear equations or a leastsquares algorithm as described above.

[0078] Alternatively, the optimization technique may determine thecontribution to the expression level of the indicator polynucleotideresulting from the homologue if the expression level of the homologue byitself is known. Such determination would not have to rely on a singleindicator polynucleotide to identify the expression level of both thetarget polynucleotide and the homologue. The optimization technique candetermine the expression level of the homologue by selecting one or more“control indicator polynucleotides” specific to the homologue. FIG. 3 isa flow diagram illustrating the overall process of correcting forhybridization of an indicator polynucleotide with a homologue of thetarget polynucleotide in one embodiment. In block 301, the techniqueselects the indicator polynucleotide. In block 302, the techniqueselects a control polynucleotide. In block 303, the technique performsthe experiment using the indicator polynucleotide and a controlindicator polynucleotide. In block 304, the technique retrieves theexpression levels from the experiment. In block 305, the techniqueadjusts the expression levels based on the characteristics of thecontrol indicator polynucleotide. The optimization techniques canidentify control indicator polynucleotides by selecting a region of the3′ untranslated of the homologue, confirming that it has no homologuesby performing a similarity search (such as a BLAST search) against adatabase of known sequences, and designing control indicatorpolynucleotide to identify that region.

[0079] The optimization technique includes this control indicatorpolynucleotide in the experiment to correct the original indicatorpolynucleotide that may be weakly identifying the homologue instead ofor in addition to the target polynucleotide. The correction may beperformed according to the following equation:

H(P _(i))=H(N _(i))+kH(N′ _(i))  (23)

[0080] where H(P_(i)) is the expression level of indicatorpolynucleotide P_(i), H(N_(i)) is the detected expression level of thetarget polynucleotide N_(i), k is a constant, and H(N′_(i)) is thedetected expression level of the homologous polynucleotide N′_(i). Theconstant k accounts for differences between the target polynucleotideand its homologue. In such a case, the contribution of the homologouspolynucleotide to the expression level measured for an indicatorpolynucleotide P_(i) will be less than its independent expression levelas measured by the control indicator polynucleotide. For example, if itis expected that the homologue will bind at ⅔ the proportion of itsactual expression level, the value of k would be ⅔. The value of k maybe determined empirically by performing hybridizations with thehomologous polynucleotide and the indicator polynucleotide whoseexpression level is the numerator of k and with the homologouspolynucleotide and the control indicator polynucleotide whose expressionlevel is the denominator of k. Alternatively it may be calculatedtheoretically using an equation such as the following equation:

H(P _(i) |N′ _(i))=f(P _(i) , N′ _(i))  (24)

[0081] where H(P_(i)|N′_(i)) is the empirically derived expression levelof polynucleotide P_(i) when hybridized to a sample containingpolynucleotide N′_(i), and f(P_(i), N′_(i)) is a function of the twopolynucleotides that are binding. The function f may take intoconsideration various factors such as the number of matches, theindividual melting temperatures of the polynucleotides assuming perfectmatches, base stacking, salt concentration, GC content, the precisepairing of mismatched bases, number of hydrogen bonds, magnesiumconcentration, primer concentration, length of perfectly matchingregions, melting temperature of perfectly matching regions, distance ofmismatches from the ends of the polynucleotides, and so on.

[0082] In the following example, the optimization technique corrects foran indicator polynucleotide that identifies a target polynucleotide andits homologue. The control indicator polynucleotide is used to determinethe independent expression level of the RNA containing the homologue.The optimization technique calculates the value of the constant k as0.723. When the experiment is run with the indicator polynucleotide andthe control indicator polynucleotide, the resulting expression levelsare 36,366 and 15,695, respectively. The optimization techniquecalculates the independent expression level of the target polynucleotideas follows:

36366=H(N _(i))+0.723*15695

H(N _(i))=36366−0.723*15695=25018

[0083] Alternatively, the optimization technique can apply the homologuecorrection as a correction matrix to matrix H, rather than matrix M.

[0084] From the foregoing, it will be appreciated that specificembodiments of the invention have been described herein for purposes ofillustration, but that various modifications may be made withoutdeviating from the spirit and scope of the invention. For example, oneskilled in the art will recognize the various ways in which thetemperature balancing method and other indicator polynucleotideselection, optimization and correction techniques described here can becombined with existing methods. For example, the indicatorpolynucleotide selection techniques may be implemented or executed bymeans of a computer. Accordingly, the invention is not limited except asby the appended claims.

I/we claim:
 1. A method for selecting indicator polynucleotides forsimultaneous detection of multiple polynucleotides in a sample, themethod comprising: identifying a plurality of indicator polynucleotides,each indicator polynucleotide having a length and a melting temperature,the melting temperature based in part on the length of the indicatorpolynucleotide; and adjusting the length of some of the identifiedindicator polynucleotides to reduce the variation in meltingtemperatures of the identified indicator polynucleotides.
 2. The methodof claim 1 wherein the selected indicator polynucleotides are used todetect polynucleotides using nucleotide array technology.
 3. The methodof claim 1 wherein the adjusting factors in the melting temperatureassociated with each portion of an identified indicator polynucleotidethat is used to detect an exon of an exon-exon junction.
 4. The methodof claim 1 wherein the selected indicator polynucleotides are used todetect splice variants.
 5. The method of claim 1 wherein the selectedindicator polynucleotides are used to detect polynucleotides frommultiple genes.
 6. The method of claim 1 wherein the adjusting attemptsto balance the melting temperature of both portions of an indicatorpolynucleotide of an exon-exon junction.
 7. A method for determining anexpression level of an exon-exon junction in a sample, the methodcomprising: selecting an indicator polynucleotide for the exon-exonjunction; determining an expected expression level resulting from theindicator polynucleotide for the exon-exon junction hybridizing to oneof the exons of the exon-exon junction that is not part of the exon-exonjunction; receiving a measured expression level for the exon-exonjunction; and calculating an estimated expression level for theexon-exon junction by adjusting the measured expression level based onthe determined expected expression level.
 8. The method of claim 7wherein the determining includes empirically deriving the expressionlevels.
 9. The method of claim 7 wherein the expression level isdetermined by performing a nucleotide array experiment with a samplethat contains an antisense polynucleotide including a portion of theselected indicator polynucleotide for the exon-exon junctioncorresponding to one of the exons and with the selected indicatorpolynucleotide for the exon-exon junction.
 10. The method of claim 7wherein the determining includes calculating a theoretical expectedexpression level.
 11. A method for identifying polynucleotides presentin a sample, the method comprising: selecting a set of indicatorpolynucleotides; identifying the corresponding indicated polynucleotidesfor the selected set and relative amounts of the indicatedpolynucleotides for the selected set that are in the sample; andadjusting the relative amounts of indicated polynucleotides based onmelting temperatures associated with the indicator polynucleotides. 12.The method of claim 11 wherein the indicator polynucleotides are used todetect splice variants.
 13. The method of claim 11 wherein the adjustingfactors in the melting temperature associated with each portion of anindicator polynucleotide that is used to detect each exon of anexon-exon junction.
 14. The method of claim 11 wherein the selected setof indicator polynucleotides includes a control indicator polynucleotideto identify relative amount of a homologue polynucleotide whose presenceis incidentally detected by another indicator polynucleotide designed todetect the presence of a target polynucleotide.
 15. The method of claim11 wherein the adjusting of the relative amounts of the indicatedpolynucleotides assumes the relative amounts vary linearly between adesired melting temperature and actual melting temperature.
 16. Themethod of claim 11 wherein the adjusting of the relative amounts of theindicated polynucleotides assumes the relative amounts vary nonlinearlybetween a desired melting temperature and actual melting temperature.17. The method of claim 11 wherein the adjusting of the relative amountsof the indicated polynucleotides is based on hybridization experimentsto determine an appropriate amount for the adjustment.
 18. A method forselecting indicator polynucleotides for simultaneous detection ofmultiple polynucleotides in a sample, the method comprising: providing aplurality of indicator polynucleotides, each indicator polynucleotidefor detecting a target polynucleotide; and when an indicatorpolynucleotide incidentally detects the presence of a polynucleotidethat is homologous to its target polynucleotide, adding a controlindicator polynucleotide whose target polynucleotide is the homologouspolynucleotide so that the amount of the target polynucleotide canfactor in the detected amount of the homologous polynucleotide.
 19. Themethod of claim 18 including performing a hybridization experiment witha homologous polynucleotide and with control indicator polynucleotide todetermining expression level for homologous polynucleotide.
 20. A methodfor selecting an indicator polynucleotide for an exon-exon junction, themethod comprising: identifying the exon-exon junction; and identifyingan indicator polynucleotide for the identified exon-exon junction sothat the melting temperature associated with the portions of theindicator polynucleotide associated with each exon of the exon-exonjunction is approximately balanced.
 21. The method of claim 20 whereinthe overall length of the indicator polynucleotide is adjusted whenbalancing the melting temperatures.
 22. The method of claim 20 whereinthe overall length of the indicator polynucleotide is not adjusted, butthe lengths of the portions are adjusted when balancing the meltingtemperatures.